CN112029823B - Metagenome library building method of nanopore sequencing platform and kit thereof - Google Patents

Abstract

The invention relates to a metagenome library construction method of a nanopore sequencing platform and a kit thereof. By optimizing and adjusting the final connection in the database construction, PCR amplification and the like, the database construction method and the kit thereof effectively reduce the initial amount of database construction, improve the reading length and quality of sequencing data, compress the time for constructing and sequencing, save the sequencing cost, improve the sensitivity of the whole process, meet the requirements of clinic on infection detection, and are suitable for popularization and application.

Description

Metagenome library building method of nanopore sequencing platform and kit thereof

Technical Field

The invention relates to the field of sequencing, in particular to a metagenome database building method based on a nanopore sequencing platform and a kit thereof.

Technical Field

Infectious diseases are a significant cause of human illness and death. Of the respiratory infections, 2016, worldwide lower respiratory infections caused at least 300 million deaths. However, due to limitations of the approach, determining the cause of lower respiratory tract infections remains challenging. Traditional methods for diagnosing lower respiratory tract infections include culture and serological examinations, with low sensitivity and long time consumption. Statistically, only 38% of adult pneumonia patients with support from imaging evidence can successfully identify the pathogen. Abuse of a broad spectrum of antibiotics makes identification of pathogens more difficult. For each patient, a slow diagnostic procedure can lead to inappropriate treatment with an empirical broad spectrum antibiotic followed by disturbances in the gut microbiome, poor treatment, prolonged hospital stays and increased cost burden. Not to mention this empiric broad spectrum antibiotic treatment can exacerbate the global multidrug resistance pathogens.

Unlike the culture method, the molecular method recognizes pathogens by abundant genetic molecules, thus skipping much time for microbial growth, and thus being much faster than the culture method. Targeted methods such as PCR are rapid diagnostic methods, but can only detect specific pathogens and are difficult to identify in a timely manner for rare or new pathogens. Therefore, rapid and accurate determination of lower respiratory tract infection pathogens is of great clinical significance.

Compared with the traditional culture identification method, the metagenome sequencing identification method has the advantages of short identification period, low requirement on the technical level of operators and the like. Metagenome sequencing also overcomes the identification defects of a plurality of microorganisms which can not be cultured, and is increasingly applied to microorganism identification, in particular to identification of pathogenic microorganisms with unknown causes. At present, the mainstream metagenome sequencing method is mainly a high-throughput sequencing method, and comprises second-generation sequencing and third-generation sequencing. The third generation of sequencing such as nanopore sequencing is a fast and convenient metagenome research method at present due to the excellent characteristics of reading the ultra-long sequence, generating and analyzing the real-time data, and being small and portable.

In the research field of metagenome, for some precious samples, the quantity of nucleic acid extracted is very limited; on the other hand, if a host removing process is used in the sample processing process, the amount of nucleic acid extracted in this way is very low, so that in many cases, in order to meet the requirements of a sequencer, it is an indispensable choice to increase the total amount of samples by PCR amplification.

At present, the existing official kits provided by Oxford nanopore company in England only comprise a rapid PCR (polymerase chain reaction) barcode kit (SQK-RPB004) and a PCR barcode kit (SQK-PBK004), wherein the official kits meet the requirements of low initial quantity and provide a multi-sample mixed sequencing function. However, the PCR Barcode kit (SQK-RPB004) adds Barcode by a transposase interruption method, which often requires complete genome, and the extracted genome is often severely fragmented for clinical samples, thereby affecting the banking success rate. The initial library building amount of the PCR barcode kit (SQK-PBK004) requires 100ng, Repair and dA tail addition are firstly carried out through a NEBNext End Repair/dA tail addition module (NEBNext End Repair/dA-tail addition module), one-step purification is carried out in the middle, a joint containing a primer site is connected to a prepared End purification product, 12 primer pairs are contained in the kit, and a PCR system (LongAmptaq 2X master mix, extension time 6mins) is carried out to amplify each sample. Then mixing the library, purifying, adding a linker, and sequencing on a computer. It can be seen that the process takes a long time, and cannot meet the clinical requirements for simple, convenient and rapid infection detection process,

the prior art still needs a metagenome sequencing database construction method which is more efficient, convenient and suitable for the low-quality characteristics of nucleic acid of clinical samples. In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The core objective to be solved by the invention is to find a sequencing library building kit which can shorten the time and save the cost. When the ONT PCR-based barcode kit (SQK-PBK004) is optimized, the invention surprisingly discovers that after the end repair enzyme and ligase of Vazyme and polymerase of Takara are selected to replace an enzyme system in the original process, the initial amount of library construction can be obviously reduced, the reading length and quality of sequencing data and the flexibility of the whole process are improved, the library construction and sequencing time is obviously shortened, and the sequencing cost is saved.

Further, the invention provides the following technical scheme:

the invention provides a library building method based on nanopore sequencing, which comprises the following steps:

step 1) DNA end repair;

step 2), connecting the BCA joints;

step 3), PCR amplification;

step 4), adding an RAP joint in a mixed library;

in some embodiments, theIn step 1), the end-repair enzyme used for end-repair is derived from Vazyme

Universal End preparation Module for

V2；

In some embodiments, the BCA linker-ligated ligase of step 2) is from Vazyme

Universal Adapter Ligation Module for

V2。

In some embodiments, the step 3) PCR amplified DNA polymerase is from Takara

GXL DNA Polymerase。

In some embodiments, the reaction system during the PCR amplification in step 3) is 50-80. mu.L, preferably 50. mu.L.

In some embodiments, the DNA tip repair of step 1) is:

components	Volume of
		Sample nucleic acid	100ng
End prep Mix4	15μL
		Nuclease-Free Water	Make up to 60 mu L

Said Endprep Mix4 comes from

Universal End preparation Module for

V2。

In some embodiments, the BCA linker system of step 2) is:

components	Volume of
		End-repair reaction product	60μL
Rapid Ligation Buffer 2	25μL
		Rapid DNA Ligase	5μL
BCA	10μL

The Rapid Ligation Buffer 2 is from

Universal Adapter Ligation Module for

V2。

In some embodiments, the PCR amplification system of step 3) is:

components	Volume of
		5×PS GXL Buffer	10μL
DNA	32.5μL
		LWB	1μL
dNTP Mix	4μL
		PrimeSTAR GXL	2μL
Nuclease-free water	Make up to 50 μ L

The 5 XPS GXL Buffer and PrimeSTAR GXL are from

GXL DNA Polymerase。

In some embodiments, the step 3) PCR amplification conditions are as follows:

in some preferred embodiments, the PCR amplification conditions of step 3) are as follows:

in some embodiments, the method is based on the method of the PCR barcode kit SQK-PBK004 of ONTs; preferably, the end repair enzymes and ligases of Vazyme and the polymerase of Takara are used to replace the enzyme system in the original scheme.

In some more preferred embodiments, a specific library construction method based on nanopore sequencing is exemplarily provided,

first, end repair of DNA fragment

1 adding an end-modification system into a PCR tube:

components	Volume of
		Sample nucleic acid	100ng
End prep Mix4	15μL
		Nuclease-Free Water	Make up to 60 mu L

2, after uniform mixing and centrifugation, carrying out PCR reaction:

temperature of	Time
		20℃	10min
65℃	10min

II, connecting a joint:

1 after the reaction is finished, adding a joint connecting system into a PCR tube:

components	Volume of
		End repair reaction product of the previous step	60μL
Rapid Ligation Buffer 2	25μL
		Rapid DNA Ligase	5μL
BCA	10μL

2, mixing evenly and incubating at 20 ℃.

Thirdly, purification

1 adding 0.4 multiplied by AMPure XP beads, uniformly mixing and incubating for 5min at room temperature, centrifuging and removing supernatant;

washing with 280% alcohol for 2 times of beads;

3 Add the nucleic-free water and incubate for 2min at room temperature.

Fourth, PCR amplification

1, preparing PCR reaction mix:

2, vortex mixing, placing on a PCR instrument, and setting a reaction program;

3 Qubit detection.

Preferably, the specific library construction method based on nanopore sequencing further comprises the following steps of purification and splicing:

fifthly, purifying and mixing the library:

1, adding 0.5 multiplied by AMPure XP beads, uniformly mixing and incubating for 5min at room temperature, centrifuging and then removing supernatant;

washing with 280% alcohol for 2 times of beads;

3, adding nucleic-free water, rotating, uniformly mixing and incubating for 2min at room temperature, and then measuring the concentration by using the Qubit;

4, mixing the libraries: and proportionally mixing the library according to the concentration of the purified sample and the data output requirement, and keeping the total amount of the mixed library at 900-.

Sixthly, adding a connector:

1 adding 0.5 xbeads, uniformly mixing at room temperature, incubating for 5min, centrifuging, and removing supernatant;

washing with 280% alcohol for 2 times of beads;

eluting with 310 mM Tris-HCl (50mM NaCl) pH 8.0 eluent, centrifuging, and detecting by using a Qubit;

4 adding 1 μ L RAP, reacting at room temperature for 5-10 min.

And seventhly, performing machine sequencing according to a standard nanopore sequencing machine flow.

The invention also provides a library construction kit for nanopore sequencing, which is based on a PCR (polymerase chain reaction) bar code kit SQK-PBK004 of ONT (optical network terminal binding protein), wherein the terminal repair enzyme in the kit is from Vazyme

Universal End preparation Module for

V2, the ligase in the kit being from Vazyme

Universal Adapter Ligation Module for

V2；

In some embodiments, the DNA polymerase in the kit is from Takara

GXL DNA Polymerase。

In some preferred embodiments, the kit comprises kit one and kit two,

wherein: the first kit comprises the following components:

the second kit comprises the following components:

in other preferred embodiments, the amounts of the components of the kit are as follows:

a kit comprises the following components (stored at minus 20 ℃ plus or minus 5 ℃):

the second kit comprises the following components (stored at 2-8 ℃):

the invention has the beneficial technical effects that:

1. according to the invention, through optimization of the last repair connection, PCR step and the like, the reading length and quality of sequencing data are improved, the sequencing time is shortened, and the sequencing cost is greatly saved.

2. The invention also effectively reduces the initial amount of library construction, can successfully construct a library for samples with extremely low initial amount of library construction, and provides possibility for sequencing detection of host-free samples and precious clinical samples.

3. The whole set of method and kit can improve the sensitivity of the whole process and increase the credibility of species identification and drug resistance analysis.

4. The invention optimizes the flow time on the whole detection flow, meets the clinical requirements on the infection detection period, shortens the whole library building time by 3 hours, has obvious significance and is suitable for popularization and application.

Drawings

Figure 13 clinical samples mz102, mz104 and mz105 were evaluated in terms of both Reads length and quality using data from two different procedures. The comparison shows that the qualified proportion of the data (part in the frame) is higher than that of the original flow after 3 samples are optimized.

Figure 23 clinical samples mz111, na103 and na113 were evaluated in terms of both Reads length and quality using data from two different procedures. The comparison shows that the qualified proportion of the data (part in the frame) is higher than that of the original flow after 3 samples are optimized.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by manufacturers, and are all conventional products available on the market.

Definition of partial terms

Unless defined otherwise below, all technical and scientific terms used in the detailed description of the present invention are intended to have the same meaning as commonly understood by one of ordinary skill in the art. While the following terms are believed to be well understood by those skilled in the art, the following definitions are set forth to better explain the present invention.

As used herein, the terms "comprising," "including," "having," and the like are used interchangeably and have the same meaning. Similarly, "comprise," "include," "have," and the like are used interchangeably and have the same meaning. In particular, each term is defined consistent with the definition of "comprising" of the common U.S. patent law, and is thus to be interpreted as an open term, meaning "at least the following," and is also to be interpreted as not excluding additional features, limitations, aspects, and the like. Thus, for example, a "device having components a, b, and c" means that the device includes at least components a, b, and c. Similarly, the phrase: by "a method involving steps a, b and c" is meant that the method comprises at least steps a, b and c. Further, although the steps and processes may be summarized herein in a particular order, skilled artisans will recognize that the ordering steps and processes may vary. Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun.

The term "about" in the present invention denotes an interval of accuracy that can be understood by a person skilled in the art, which still guarantees the technical effect of the feature in question. The term generally denotes a deviation of ± 10%, preferably ± 5%, from the indicated value.

Furthermore, the terms first, second, third, (a), (b), (c), and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

The term "nucleic acid" in the present invention may refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single-or double-stranded form. The term can encompass nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, have similar binding properties as the reference nucleic acid, and are metabolized in a manner similar to the reference nucleotides. Examples of such analogs can include, but are not limited to, phosphorothioates, phosphoramidites, methyl phosphonates, chiral methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs). The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.

The term "nucleotide" in the context of the present invention, in addition to a naturally occurring ribonucleotide or deoxyribonucleotide monomer, is understood to refer to structurally related variants thereof, including derivatives and analogs, that are functionally equivalent with respect to the particular environment in which the nucleotide is used (e.g., hybridization to a complementary base), unless the context clearly dictates otherwise.

The term "primer" in the present invention may refer to a short nucleic acid sequence that provides a starting point for DNA synthesis.

The term "nanopore" in the present invention refers to a pore, channel, or passage formed or otherwise provided in a membrane. The membrane may be an organic membrane, such as a lipid bilayer, or a synthetic membrane, such as a membrane formed from a polymeric material. The nanopore may be adjacent or proximate to or coupled to a sensing circuit, such as, for example, an electrode arrangement of a Complementary Metal Oxide Semiconductor (CMOS) or Field Effect Transistor (FET) circuit. In some examples, the nanopore has a characteristic width or diameter of about 0.1 nanometers (nm) to about 1000 nm. Some nanopores are proteins.

The term "nanopore sequencing" or "nanopore-based sequencing" in the present invention refers to a method of determining the sequence of a polynucleotide by means of a nanopore. In some embodiments, the sequence of the polynucleotide is determined in a template-dependent manner. The methods disclosed herein are not limited to any nanopore sequencing method, system, or device.

The term "barcode" in the present invention means an oligonucleotide present in a nucleic acid sequence in order to identify it.

The term "sequencing" in the context of the present invention refers to the determination of the order and position of bases in a nucleic acid.

Reagents and consumables used in the invention:

nucleic acid purification reagents: AgencourtAmpure XP beads (cat # A63881); enzyme-free sterile water: ThermoFisher, nucleic-Free Water (not DEPC-Treated) (Cat: AM 9937); DNA detection kit of the Qubit fluorescence quantitative instrument: qubit 1X dsDNA HS Assay Kit (cat # Q33231); a database building end repairing module:

Universal End preparation Module for

v2 (cat # N203); a database building end repairing module: NEBNext Ultra II End repair/dA-tailing Module (cat # E7546); building a warehouse and connecting a connecting module:

Universal Adapter Ligation Module for

v2 (cat # N204); building a warehouse and connecting a connecting module: NEB Blunt/TA Ligase Master Mix (cat # M0367); constructing a library PCR amplification enzyme: of Takara

GXL DNA Polymerase (cat # R050A); constructing a library PCR amplification enzyme:

taq DNA Polymerase (cat # M0323L); constructing a library PCR amplification enzyme: nucleic acid standard Zymobiomics (cat # KK2602) of HifiHotstart Ready mix (cat # KK2602)^TM Microbial Community DNA Standard(Catalog Nos.D6305)。

Rapid PCR barcode kit (SQK-RPB004) of ONT, oxford nanopore inc, uk: FRM (fragmentation mixture): 1, pipe; RLB (fast barcode primer): 01-12A, 12 tubes in total; RAP (quick joint): 1, pipe; SQB (sequencing buffer): 1, pipe; SQT (sequencing membrane immobilizates): 1, pipe; LB (sequencing loading beads): 1 tube.

PCR barcode kit of ONT of Oxford nanopore Inc. England (SQK-PBK 004): LWB (barcode primer): 01-12, 12 tubes in total; RAP (quick joint): 1, pipe; SQB (sequencing buffer): 1, pipe; LB (sequencing loading beads): 1 tube.

Example 1 Performance optimization and parameter adjustment experiments

The invention carries out optimization experiment based on PCR bar code kit (SQK-PBK004) of ONT.

The invention firstly selects a DNA nucleic acid standard substance Zymobiomics produced by Zymo Research company^TMThe Microbial Community DNA Standard (CatalogNos. D6305) was used as the input for the validation, which consisted of the precise quantitative ratioThe total GC content distribution is very broad (15% -85%, table below, DNA standard composition (Catalog nos. d6305, product specification)). it is a good standard for the study of microbiome.

TABLE 1 ZymoBIOMICS^TMMicrobial Community DNA Standard Strain information

1) Optimized screening for end repair and ligase

In consideration of high requirements of metagenome library construction sequencing on sample size, experiment precision, time cost and the like, enzyme systems of different models have great influence on library construction or sequencing results in the experimental process. Therefore, in the early stage of the invention, several enzymes such as an end repair-ligation module (cat No. KK8511), a Tiangen TIANCANSeq rapid DNA fragmentation/end repair/dA addition module (cat No. NG301), a Xianseng rapid end repair/A tail addition module (cat No. 12605ES24) and the like in KAPA Hyperplus kit are pre-screened to obtain the end repair and ligation reagent of the domestic Vazyme of Nanjing noprazan organism Vazyme with better effect

Universal End preparation Module for

V2 (cat # N203) and

Universal Adapter Ligation Module for

v2 (cat # N204).

Furthermore, the invention uses ZymoDNA standard as input sample, and comparatively tests

Universal End preparation Module for

V2 (cat # N203) and

Universal Adapter Ligation Module for

the final repair-ligation effect of V2 (cat # N204) with ONT official reagents NEBNext Ultra II End repair/dA-ligation Module (cat # E7546) and NEB Blunt/TA Ligase Master Mix (cat # M0367).

As shown in the following table, surprisingly, the end-repair-ligation process of the Vazyme reagent produced in China always takes 30min shorter than the NEB reagent recommended by ONT official, and the final concentration of the nucleic acid output from the PCR process is higher than that of the NEB by using the same PCR process regardless of the lower (1ng) or lower (0.1ng) initial amount of the library, which proves that the Vazyme reagent has shorter process time and better end-repair-ligation effect.

2) Optimized screening for DNA polymerases

In order to further optimize the reagents for the PCR step, the present invention still utilizes Zymo DNA standards and performs the same end-of-line ligation protocol. In addition to ONT officials using NEB's LongAmp Hot Start Taq 2X Master Mix (cat # M0533S), the present invention also contemplates the co-testing of products from several other companies, such as Takara

GXL DNA Polymerase (cat # R050A) and HifiHotstart Ready mix of KAPA (cat # KK 2602).

As shown in the following table, the PCR procedure of Takara takes a minimum time (54min) and the amplification rate is 1000bp per synthesisIt took 10s, while both the other enzymes took 1 min. And the product yield after the PCR of the Takara enzyme is the highest; the enzyme KAPA was obtained in good yield, but the process time was still relatively long (130 min). As can be seen, the polymerase selects for Takara

GXL DNA Polymerase (cat # R050A).

3) Use condition-optimized screening of DNA polymerases

Next, the present invention is directed to Takara

The specific conditions of use (including annealing temperature, PCR reaction volume, extension time) of GXL DNA Polymerase (cat # R050A) were further optimized. First, as shown in the following table, the present invention tested two annealing temperatures (56 ℃ C. and 65 ℃ C.) and two reaction systems (50. mu.L, 80. mu.L, and 100. mu.L), respectively, and the concentration was determined after the PCR reaction was completed, and the concentration of the amplification product could roughly evaluate the PCR amplification yield, and it was found that the conditions for annealing at 65 ℃ in the 50. mu.L system were optimal. Then, 500ng of DNA was taken for subsequent purification steps, since the present invention used 0.5 × beads purification, and some smaller fragments, such as primer dimers, which are not targets of the present invention, were discarded. Therefore, the purification yield can be more accurately measured as the amplification efficiency of PCR, and from this index, the conditions of 50. mu.L system and 65 ℃ annealing are also optimal.

Then, the present invention further evaluated the extension time, in order to obtain a more optimal amplification length, the extension time of 45-60s, preferably 45s, was finally selected by the test shown in the following table, resulting in an optimal purification yield, and the average length of the amplified fragments was also optimal by sequencing evaluation.

Example 2 establishment of the Process System according to the invention

Based on the optimization experiment of example 1, the library construction methodology of the present invention was obtained, which was based on the PCR barcode kit (SQK-PBK004) for ONT.

First, end repair of DNA fragment

1 Add the end-modification system to a 0.2mL PCR tube:

note: the total amount of the sample extracted was made up to 100ng and 45. mu.L of the sample was added.

2, lightly blowing and uniformly mixing by using a pipettor (do not shake and uniformly mixing), collecting reaction liquid to the bottom of the tube after short-time centrifugation, and placing the PCR tube on a PCR instrument to perform the following reactions:

temperature of	Time
		20℃	10min
65℃	10min

II, connecting a joint:

1 after the reaction is finished, adding a joint connecting system into a PCR tube:

2, the mixture is blown and beaten by a pipette and is mixed evenly, a PCR tube is placed on a PCR instrument, and the mixture is incubated for 15min at the temperature of 20 ℃.

Thirdly, purification

1 adding 0.4 multiplied by AMPure XP beads, rotating, mixing uniformly and incubating for 5min at room temperature, placing on a magnetic frame for standing after short-time centrifugation, and removing supernatant;

2, washing the newly prepared 80% alcohol with 200 mu L of beads for 2 times, and injecting: 80% alcohol needs to be prepared at present.

3 adding 35 mu L of nucleic-free water, uniformly mixing and incubating for 2min at room temperature, centrifuging for a short time, and putting on a magnetic frame until the mixture is clear;

4 carefully pipette 33.5. mu.L of the supernatant into a 0.2mL PCR tube, and 1. mu.L is taken for the concentration determination of the Qubit.

Fourth, PCR amplification

1 PCR reaction mix was prepared in 0.2mL PCR tubes:

2, uniformly mixing by vortex, collecting reaction liquid to the bottom of the tube after short-time centrifugation, and placing the PCR tube on a PCR instrument to set a reaction program;

3 PCR is finished, and the concentration of the Qubit is detected and recorded.

Fifthly, purification

1, adding 0.5 multiplied by AMPure XP beads into a PCR reaction tube, uniformly rotating and incubating for 5min at room temperature, placing on a magnetic frame after short-time centrifugation, and removing supernatant after liquid is clarified;

2 washing 200 μ L of freshly prepared 80% alcohol for 2 times of beads;

3 adding 26 mu L of nucleic-free water, uniformly mixing and incubating for 2min at room temperature, centrifuging for a short time, and placing on a magnetic frame until the mixture is clear;

4 carefully pipette 25. mu.L of the supernatant into a 1.5mL centrifuge tube, and measure the concentration with 1. mu.L of the Qubit.

Sixthly, mixed storage: and proportionally mixing the library according to the concentration of the purified sample and the data output requirement, and keeping the total amount of the mixed library at 900-.

Seventhly, purifying and adding a joint:

1 adding 0.5 xbeads, uniformly mixing at room temperature, incubating for 5min, centrifuging for a short time, and removing supernatant on a magnetic frame;

2 washing the beads with 200 μ L of freshly prepared 80% alcohol for 2 times;

3 adding 12 μ L10 mM Tris-HCl (50mM NaCl) pH 8.0 eluent, rotating and mixing uniformly at room temperature, incubating for 2min, centrifuging for a while, and placing on a magnetic frame until the solution is clear; carefully pipette 11. mu.L of the supernatant into 1.5mL of a low-adsorption centrifuge tube;

4 QC: detecting by taking 1 mu LQubit;

5, adding a joint: mu.L RAP was added to the above 10. mu.L sample and reacted at room temperature for 5-10 min.

And eighthly, performing machine sequencing according to a standard nanopore sequencing machine flow.

EXAMPLE 3 preparation of the kit

According to the flow system determined in example 2, we assemble the scheme into 2 library preparation kits as shown in the following table, and in view of the storage temperature difference of the kits, the kits are prepared into two packaged kits which need to be stored under the conditions of minus 20 ℃ +/-5 ℃ and 2-8 ℃.

A kit comprises the following components (stored at minus 20 ℃ plus or minus 5 ℃):

the second kit comprises the following components (stored at 2-8 ℃):

example 4 clinical sample testing-comparison with SQK-PBK004

Based on the kit of the invention in the embodiment 3, the invention utilizes 6 clinical sputum nucleic acid samples, and respectively builds the libraries and machines by the original process of the PCR barcode kit (SQK-PBK004) and the current process of the embodiment 2 for comparison with different dimensions.

Firstly, as shown by comparison of database building time of the original process and the current process in the table, most of the database building steps are reduced after optimization, the time of the whole process is shortened by 3 hours, and the requirement of clinical rapidness for infection detection products can be greatly met.

	Current process (min)	Original process (min)
			Tip repair	25	53
Connection of	58	73
			PCR	54	191
Mix storehouse and operate machine	93	93
			Total length of time	230	410

Further, by the on-machine sequencing, the invention analyzes the proportion of quality control filtration according to the aspects of both the masses of Reads and the lengths of Reads in each sample sequencing data, and the proportion of Reads which is finally aligned to the microorganisms and can be used for species identification. As shown in the following table and in fig. 1 and 2, the present flow has significant advantages both in terms of data quality and in terms of Reads length, in particular in terms of Reads length. The original process generally has a short Reads length and is largely filtered. Thus, the final available proportion of microorganisms Reads is now higher than the original flow for 6 samples. The method can not only compress sequencing time and save sequencing cost, but also improve the sensitivity of the process, and greatly increase the reliability of species identification and drug resistance analysis.

Example 5: clinical sample testing-comparison with SQK-RPB004

To further evaluate the advantages of the kit of the present invention, this example compares the effects of the kit of the present invention and another one of the ONT banking kits SQK-RPB 004. From the process given by the official, the minimum initial amount of the library construction of SQK-PBK004 is 100ng, and the minimum initial amount of the library construction of SQK-RPB004 is 1ng, and in order to compare the library construction success rates of the library construction kit and the SQK-RPB004 kit of the invention on samples with low initial amounts, the invention designs the following experiments.

The invention carries out gradient dilution on nucleic acid extracted from the same clinical alveolar lavage fluid sample, takes different database construction initial quantities to carry out database construction machine sequencing of SQK-PBK004 and SQK-RPB004 kits in parallel, and finally compares the ratio of Reads on microbial genomes as shown in the following table, and the database construction is maintained to be more than 20% by using the current process, even if the database construction initial quantity is extremely low (8pg), the current process can meet the condition that the nucleic acid extraction concentration of a host-free sample and a precious clinical sample is low. And the ratio of Reads of the SQK-RPB004 library building computer data with the same initial amount compared with the SQK-PBK004 process with the same initial amount of the library building is very low, and the integral comparison Reads number does not form a gradient with the initial amount of the library building, which indicates that the stability of the whole process is poor. In addition, the number of Reads detected by the SQK-PBK004 process is more from the aspect of pathogen detection, and the reliability of detection and drug resistance is increased. In conclusion, the optimized SQK-PBK004 process (namely the kit disclosed by the invention) can be used for successfully constructing a library for samples with extremely low initial library construction amount, detecting infection pathogens and providing possibility for trace nucleic acid sequencing detection of host samples, precious clinical samples and the like.

The above description of the specific embodiments of the present application is not intended to limit the present application, and those skilled in the art may make various changes and modifications according to the present application without departing from the spirit of the present application, which is intended to fall within the scope of the appended claims.

Claims (9)

1. A method for creating a library based on nanopore sequencing, the method comprising the steps of:

step 1) DNA end repair;

step 2), connecting the BCA joints;

step 3), PCR amplification;

step 4), adding an RAP joint in a mixed library;

in the step 1), the end-repairing enzyme used for end-repairing is derived from Vazyme

Universal End preparation Module for

V2; the BCA linker-ligated ligase in step 2) is derived from Vazyme

Universal Adapter Ligation Module for

V2; said step 3) PCR amplified DNA polymerase is from Takara

GXL DNA Polymerase；

The method is based on a library building method of a PCR bar code kit SQK-PBK004 of ONT.

2. The method for constructing a library based on nanopore sequencing according to claim 1, wherein the reaction system in the PCR amplification process in step 3) is 50-80 μ L.

3. The method for constructing a library based on nanopore sequencing according to claim 2, wherein the reaction system in the PCR amplification process of step 3) is 50 μ L.

4. The method for library construction based on nanopore sequencing according to any of claims 1-3, wherein the DNA tip repair system of step 1) is:

components Volume of Sample nucleic acid 100ng End prep Mix4 15μL Nuclease-Free Water Make up to 60 mu L

The End prep Mix4 is from

Universal End preparation Module for

V2。

5. The method for library construction based on nanopore sequencing according to claim 4, wherein the BCA linker system of step 2) is:

components Volume of End-repair reaction product 60μL Rapid Ligation Buffer 2 25μL Rapid DNA Ligase 5μL BCA 10μL

The Rapid Ligation Buffer 2 is from

Universal Adapter Ligation Module for

V2。

6. The method for library construction based on nanopore sequencing according to any one of claims 1-3, wherein the PCR amplification system of step 3) is:

components Volume of 5×PS GXL Buffer 10μL DNA 32.5μL LWB 1μL dNTP Mix 4μL PrimeSTAR GXL 2μL Nuclease-free water Make up to 50 μ L

The 5 XPS GXL Buffer and PrimeSTAR GXL are from

GXL DNA Polymerase。

7. The method for constructing a library based on nanopore sequencing according to claim 6, wherein the PCR amplification conditions of step 3) are as follows:

8. a kit for preparing a library for nanopore sequencing, wherein a terminal repair enzyme in the kit is derived from Vazyme

Universal End preparation Module for

V2, the ligase in the kit being from Vazyme

Universal Adapter Ligation Module for

V2, the DNA polymerase in the kit being from Takara

GXL DNA Polymerase; the kit is based on a PCR bar code kit SQK-PBK004 of ONT.

9. The pooling kit for nanopore sequencing of claim 8, wherein said kit comprises a kit one and a kit two component, wherein:

the kit comprises the following components:

the kit comprises the following two components:

Images